The present invention relates to novel conjugated forms of pharmacologically active agents, and methods for the preparation and use thereof. In a particular aspect of the invention, methods are provided for simultaneously treating a pathological condition with a pharmacologically active agent and reducing nitric oxide levels.
Despite the advent of modem pharmaceutical technology, many drugs still possess untoward toxicities which often limit the therapeutic potential thereof. For example, although non-steroid anti-inflammatory drugs (NSAIDs) are a class of compounds which are widely used for the treatment of inflammation, pain and fever, NSAIDs (e.g., aspirin, ibuprofen and ketoprofen) can cause gastrointestinal ulcers, a side-effect that remains the major limitation to the use of NSAIDs (see, for example, J. L. Wallace, in Gastroenterol. 112:1000-1016 (1997); A. H. Soll et al., in Ann Intern Med. 114:307-319(1991); and J. Bjarnason et al., in Gastroenterol. 104:1832-1847 (1993)).
There are two major ulcerogenic effects of NSAIDs: (1) topical irritant effects on the epithelium of the gastrointestinal tract and (2) suppression of gastrointestinal prostaglandin synthesis. In recent years, numerous strategies have been attempted to design and develop new NSAIDs that reduce the damage to the gastrointestinal tract. These efforts, however, have largely been unsuccessful. For example, enteric coating or slow-release formulations designed to reduce the topical irritant properties of NSAIDs have been shown to be ineffective in terms of reducing the incidence of clinically significant side effects, including perforation and bleeding (see, for example, D. Y. Graham et al., in Clin. Pharmacol. Ther. 38:65-70 (1985); and J. L. Carson, et al., in Arch. Intern. Med., 147:1054-1059 (1987)).
It is well recognized that aspirin and other NSAIDs exert their pharmacological effects through the inhibition of cyclooxygenase (COX) enzymes, thereby blocking prostaglandin synthesis (see, for example, J. R. Van in Nature, 231:232-235 (1971)). There are two types of COX enzymes, namely COX-1 and COX-2. COX-1 is expressed constitutively in many tissues, including the stomach, kidney, and platelets, whereas COX-2 is expressed only at the site of inflammation (see, for example, S. Kargan et al. in Gastroenterol., 111:445-454 (1996)). The prostagladins derived from COX-1 are responsible for many of the physiological effects, including maintenance of gastric mucosal integrity.
Many attempts have been made to develop NSAIDs that only inhibit COX-2, without impacting the activity of COX-1 (see, for example, J. A. Mitchell et al., in Proc. Natl. Acad. Sci. USA 90:11693-11697 (1993); and E. A. Meade et al., in J. Biol. Chem., 268:6610-6614 (1993)). There are at least two NSAIDs presently on the market (i.e., nabumetone and etodolac) that show marked selectivity for COX-2 (see, for example, E. A. Meade, supra.; and K. Glaser et al., in Eur. J. Pharmacol. 281:107-111 (1995)). These drugs appear to have reduced gastrointestinal toxicity relative to other NSAIDs on the market.
On the basis of encouraging clinical as well as experimental data, the development of highly selective COX-2 inhibitors appears to be a sound strategy to develop a new generation of anti-inflammatory drugs. However, the physiological functions of COX-1 and COX-2 are not always well defined. Thus, there is a possibility that prostagladins produced as a result of COX-1 expression may also contribute to inflammation, pain and fever. On the other hand, prostagladins produced by COX-2 have been shown to play important physiological functions, including the initiation and maintenance of labor and in the regulation of bone resorption (see, for example, D. M. Slater et al., in Am. J. Obstet. Gynecol., 172:77-82 (1995); and Y. Onoe et al., in J. Immunol. 156:758-764 (1996)), thus inhibition of this pathway may not always be beneficial. Considering these points, highly selective COX-2 inhibitors may produce additional side effects above and beyond those observed with standard NSAIDs, therefore such inhibitors may not be highly desirable.
Since anthracyclines such as adriamycin are commonly used antitumor agents, considerable efforts have also been made to develop strategies for reducing the acute and delayed cardiomyopathies induced by anthracyclines, while maintaining the therapeutic efficacy of these compounds. The molecular mechanism of cardiomyopathy is now attributed to the adriamycin-induced release of iron from intracellular iron proteins, resulting in the formation of an adriamycin-iron complex. The adriamycin-iron complex generates reactive oxygen species, causing the scission and condensation of DNA, peroxidation of phospholipid membranes, depletion of cellular reducing equivalents, interference with mitochondrial respiration, and disruption of cell calcium homeostasis (see, for example, Myers et al., in Science 197:165-167 (1977); and Gianni et al., in Rev. Biochem. Toxicol. 5:1-82 (1983)). In addition to cardiomyopathy, adriamycin administration causes cutaneous irritation and alopecia, mucositis (stomatitis and esophagitis), phlebosclerosis and hematologic toxicities and many other local and systemic toxicities.
Recently, ICRF-187 (i.e., dexrazoxane) has been demonstrated to be effective for the removal of iron from the anthracycline-iron complex, therefore preventing the cardiac toxicity in cancer patients receiving adriamycin chemotherapy (see, for example, Kolaric et al., in Oncology 52:251-5 (1995)). However, when chelated with iron, the iron-ICRF-187 complex per se is also very effective at promoting hydroxyl radical generation via the Fenton reaction, causing oxidative damage to tissues (see, for example, Thomas et al., in Biochem. Pharmacol., 45:1967-72 (1993)). In addition, since ICRF-187 is a strong chelator (having a structure similar to EDTA), it chelates not only low-molecular-weight iron, but also chelates iron from transferrin and ferritin, as well as copper from ceruloplasmin, thus potentially affecting normal cellular iron metabolism.
Accordingly, there is still a need in the art for modified forms of NSAIDs, and other pharmacologically active agents, which cause a reduced incidence of side-effects, relative to the incidence of side-effects caused by such pharmacologically active agents as aspirin, ibuprofen, and the like.
In accordance with the present invention, there are provided conjugates of physiologically compatible nitric oxide scavengers (e.g., dithiocarbamates (DC)) and pharmacologically active agents (e.g., NSAIDS). Invention conjugates provide a new class of pharmacologically active agents (e.g., anti-inflammatory agents) which cause a much lower incidence of side-effects due to the protective effects imparted by modifying the pharmacologically active agents as described herein.
Recent evidence has shown that NSAID administration up-regulates the expression of inducible nitric oxide synthase (see, for example, B. J. R. Whittle et al., in Br. J. Pharmacol., 116:2286-2290 (1995)). Excessive nitric oxide produced from inducible nitric oxide synthase is known to contribute to the generation of mucosal damage (see, for example, S. J. Middleton et al., in Lancet 341:456-466 (1993); and M. J. S. Miller et al., in Scand. J. Gastroenterol., 264:11-16 (1993)). When chelated with iron (e.g., intracellular iron), nitric oxide scavengers (such as a dithiocarbamate-iron complex) becomes an effective nitric oxide scavenger which binds tightly to nitric oxide and reduces in vivo nitric oxide levels. It is now recognized that excessive nitric oxide production can induce the expression of COX-2, thereby enhancing the cascade of inflammatory reactions. Thus, scavenging NO by a nitric oxide scavenger (such as the dithiocarbamate-iron complex) could reduce the negative consequences brought about by excessive COX-2 levels, by reducing the expression of COX-2.
In summary, there are a number of advantages of conjugates according to the invention (e.g., DC-NSAID), including:
(i) reduced topical irritant effects of NSAIDs,
(ii) enhanced tissue delivery of both drugs as a result of a decrease in net charges on the molecule, particularly for acidic NSAIDs such as aspirin, diclofenac and ibuprofen, thereby reducing the quantity of material which must be delivered to achieve an effective dosage,
(iii) chelating intracellular free iron ions, thereby preventing iron-related oxidative damage,
(iv) inhibiting VCAM-1 expression, thereby blocking neutrophil adherence to the vascular endothelium induced by NSAID administration, and
(v) scavenging intracellular nitric oxide, thereby preventing the production of peroxynitrite, a potent oxidant, and reducing the induction of COX-2 expression, which could induce further inflammatory response.
In another aspect of the invention, there are described bio-cleavable conjugates of a suitable nitric oxide scavenger (e.g., dithiocarbamate) and an anti-neoplastic agent (e.g., adriamycin, wherein the resulting conjugate is referred to as DC-adriamycin), which alleviate some of the toxicities associated with administration of anti-neoplastics such as adriamycin. There are a number of advantages of DC-adriamycin over adriamycin alone, including:
(i) reducing cutaneous irritation and alopecia and vascular damage (because the conjugates are inactive until they have reached the intracellular site of action),
(ii) chelating intracellular iron, thus reducing free radical-induced acute and delayed cardiomyopathies, and
(iii) removing excessive nitric oxide produced from malignant and cancerous tissues.